ABSTRACT Glioblastoma (GBM) is the most aggressive brain tumor, and has a median survival of only 12-15 months despite intensive therapies, indicating the urgent need to identify effective approaches to treat GBM and circumvent resistance to therapies in order to significantly improve patient survival. Our recent studies demonstrated that lipid synthesis and uptake are greatly enhanced in GBM and promote tumor growth. It is known that increased free fatty acids (FFA) and cholesterol can cause endoplasmic reticulum (ER) stress and lipotoxicity that lead to cell death, which raises the intriguing question of how GBM cells can prevent the toxicity potentially induced by increased lipid metabolism. We recently found that tumor tissues from GBM patients contain large amount of lipid droplets (LDs), triglycerides (TG) and cholesteryl esters (CE), suggesting that GBM cells may store excess FFA and cholesterol into LDs to avoid toxicity and maintain tumor growth. Further analysis showed that GBM patients whose tumor tissues contained higher levels of LDs and of DGAT1 (diglyceride acyltransferase) or SOAT1 (sterol O-acyltransferase), two ER membrane-bound enzymes that catalyze the conversion of excess FFA and cholesterol into TG and CE to form LDs, had the worse survival, suggesting that LDs may have a protumoral function. Consistent with our findings, several groups have recently reported that inhibiting cholesterol esterification or suppressing LD formation in prostate, pancreatic or renal cancer cells significantly enhanced ER stress. Nevertheless, many important LD functions in cancer cells remain unexplored, such as their energetic role and potential for supporting tumor resistance. Our preliminary data suggest that LDs may provide a critical energy source for GBM survival under nutrient reduction or radiation/temozolomide (TMZ) treatment, the standard therapy for GBM. Based on current understanding of LDs in cancer cells and our novel preliminary data, we hypothesize that LD formation in GBM prevents ER stress and lipotoxicity, and also serves as energy reservoir to support tumor survival upon energy challenges. We further hypothesize that inhibiting LD formation will cause ER stress, lipotoxicity and energy shortage, which may strongly synergize with radiation/TMZ treatment to induce GBM cell death. In this study, we will: (1) delineate the underlying protective role of LD formation in GBM cells; (2) determine whether LDs play an important energetic role in GBM cells; (3) examine whether genetically or pharmacologically inhibiting LD formation effectively suppresses tumor growth and sensitizes GBM to radiation/TMZ treatment in GBM orthotopic mouse models. This study will reveal the previously uncharacterized role and molecular regulation of LDs in GBM. Importantly, it will also demonstrate that inhibiting LD formation may be a very effective approach to specifically target GBM with little toxicity on normal brain tissues where no LDs could be detected. Completion of this study will significantly advance our understanding of lipid metabolism reprogramming and may bring about new approaches to antagonize GBM.